This invention generally relates to imaging moving objects or particles for purposes of analysis and detection, and more specifically, to a system and method for determining and analyzing the morphology of moving objects, such as cells, and for detecting the presence and composition of Fluorescence In-Situ Hybridization (FISH) probes within cells.
There are a number of biological and medical applications that are currently impractical due to limitations in cell and particle analysis technology. Examples of such biological applications include battle field monitoring of known airborne toxins, as well as the monitoring of cultured cells to detect the presence of both known and unknown toxins. Medical applications include non-invasive prenatal genetic testing and routine cancer screening via the detection and analysis of rare cells (i.e., low rate of occurrence) in peripheral blood. All of these applications require an analysis system with the following principal characteristics:
1. high speed measurement;
2. the ability to process very large or continuous samples;
3. high spectral resolution and bandwidth;
4. good spatial resolution;
5. high sensitivity; and
6. low measurement variation.
In prenatal testing, the target cells are fetal cells that cross the placental barrier into the mother""s bloodstream. In cancer screening, the target cells are sloughed into the bloodstream from nascent cancerous tumors. In both of these applications of this technology, the target cells may be present in the blood at concentrations of one to five cells per billion. This concentration yields approximately 20-100 target cells in a typical 20 ml blood sample. The extreme rarity of the targeted cells demands that any detection and analysis system employed in these applications be capable of processing an enriched sample of approximately 100 million cells within a few hours, corresponding to a minimum throughput of 10,000 cells per second. Cell processing includes the determination of cellular morphology parameters such as overall size, nuclear size, nuclear shape, and optical density, the detection and characterization of numerous fluorescent markers and FISH probes, the quantification of the total amount of DNA in the nucleus, and the detection of other cellular components such as fetal hemoglobin. To accomplish these processing tasks, the system must be able to collect cell images with a spatial resolution of approximately 1 micron. Likewise, the system must have high spectral resolution and bandwidth to differentiate four or more fluorescent colors. Since some probes may label important cellular features with only a few thousand fluorescent molecules, the system must have high sensitivity and good measurement consistency to differentiate very weak signals.
The predominant research laboratory protocols for non-invasive prenatal diagnosis employ a complex series of process steps that include gradient centrifugation to remove unnucleated cells, high-speed cell sorting for fetal cell enrichment, and fluorescence microscopy for fetal cell identification and genetic analysis. These protocols often yield little or no fetal cells for analysis, because a fraction of the fetal cells are lost at each step of the protocol. Nevertheless, the protocols cannot be simplified because of limitations in existing analysis technology. Ideally, fetal cell identification and analysis would be performed in a few hours by a high-speed cell sorter having the necessary speed and sample handling capacity. This ideal is not possible with conventional systems, because conventional cell sorters lack the necessary imaging abilities, sensitivity, and repeatability to reliably identify fetal cells and enumerate the number and color of FISH probes used to make the diagnosis. Therefore, under current protocols, cells must be sorted onto slides and examined using fluorescence microscopy to establish their fetal origin and make a genetic diagnosis. The combination of low fetal cell yields and lengthy processing times precludes the clinical application of non-invasive fetal testing with existing technology.
No technology prior to the present invention incorporates all six of the principal characteristics of a viable fetal cell or cancer analysis system. In the prior art, there have been advances that might be applied to these applications, but significant limitations still remain.
A paper published by Ong et al. [Anal. Quant. Cytol. Histol., 9(5):375-82] describes the use of a time-delay and integration (TDI) detector in an imaging flow cytometer. A TDI detector is any pixilated device in which the signal produced in response to radiation directed at the device can be caused to move in a controlled fashion. Typically, the pixels of a TDI detector are arranged in rows and columns, and the signal is moved from row to row in synchrony with a moving image projected onto the device, allowing an extended integration time without blurring. The approach disclosed by Ong et al. advanced the art by addressing the need for spatial resolution and high sensitivity for cells in flow. However, this approach does not address the remaining principal characteristics. The authors of this paper cite an operating speed of 10 cells per second and a theoretical speed limitation of 500 cells per second, which is at least an order of magnitude slower than is required for non-invasive fetal testing. In addition, the system has no spectral resolution; laser scatter and fluorescent light are collected by the imaging system indiscriminately.
In more recent developments, U.S. Pat. No. 5,644,388 discloses an alternative approach to an imaging flow cytometer. The patent discloses the use of a frame-based image collection approach in which a video camera views cells in flow, in a freeze frame fashion. This method requires the image collection system to be synchronized with the presence of cells in the imaging area, unlike the case of TDI, wherein the detector readout rate is synchronized with the velocity of the cells. When a cell is imaged with the frame-based method, the integration period must be very short to prevent blurring. A short integration time is achieved either with a strobed light source, or a continuous light source combined with a shuttered detector. In either case, the short integration time reduces the signal-to-noise ratio and the ultimate sensitivity of the approach. Further, frame-based cameras require time to transfer data out of the camera, during which no images are acquired, and cells of interest can escape detection. Finally, like the work of Ong et al., this patent makes no provisions for acquiring data over a large spectral bandwidth and with sufficient spectral resolution to simultaneously resolve numerous and differently colored fluorescent probes and FISH spots.
Spectral discrimination is addressed in U.S. Pat. No. 5,422,712, in which the spectra of particles suspended in a fluid are collected as the particles flow through a detection region. However, there is no spatial representation of the object in the system disclosed in this patent, because the object is defocussed at the detector. In this system, light is collected from the object and an image is created at an intermediate aperture. The light continues through the aperture to a spectral dispersing element, which disperses the light spectrally along the axis of flow. The dispersed light is applied to an image intensifier in which it is amplified, and the light signal output from the image intensifier is finally directed to frame-based detector. At the intermediate aperture, prior to spectral dispersion, the image represents the spatial distribution of light in object space. The spatial distribution is blurred as the light propagates past the image plane, through the spectral dispersing element and onto the image intensifier. Because there is no provision for re-imaging the intermediate aperture at the intensifier, the resulting signal distribution at the intensifier represents only the spectral distribution of the light and does not preserve the spatial distribution of the light from the object. The loss of spatial information limits the utility of the invention for applications such as fetal cell analysis. If multiple identical FISH spots are present in a cell, their spectra can be ascertained using this approach, but the number of spots cannot be determined. In addition, this approach disperses the wavelength spectrum parallel to the axis of flow. If two particles are illuminated in the flow axis, their spectra can overlap on the detector. To prevent this problem, the patent discloses that a very short illumination height in the flow axis is used. The short illumination height decreases integration time, which necessitates the use of the image intensifier. Further, the short illumination height limits throughput by preventing the simultaneous imaging of multiple cells in the flow axis.
Accordingly, it will be apparent that an improved technique is desired that resolves the limitations of the conventional approaches discussed above. It is expected that the new approach developed to address these problems in the prior art will also have application to the analysis of other types of moving objects besides cells and may be implemented in different configurations to meet the specific requirements of disparate applications of the technology.
The present invention is directed to an imaging system that is adapted to determine one or more characteristics of an object from an image of the object in a broad, flat flow. There is relative movement between the object and the imaging system, and although it is contemplated that either (or both) may be in motion, the object will preferably move while the imaging system will be fixed. In addition, it should also be understood that while much of the following summary and the corresponding claims recite xe2x80x9can object,xe2x80x9d it is clearly contemplated that the present invention is preferably intended to be used with a plurality of objects and is particularly useful in connection with imaging a stream of objects in a broad, flat flow.
The present invention provides a method and apparatus for the analysis of rare cells in the blood for the purposes of non-invasive fetal cell diagnosis and cancer screening, as well as other applications. To achieve such functions, the present invention is capable of rapidly collecting data from a large cell population with high sensitivity and low measurement variation. These data include simultaneous spatial and spectral images covering a large bandwidth at high resolution. Further, the present invention preserves the spatial origin of the spectral information gathered from the object.
Several different embodiments of the imaging system are provided. One preferred form of the invention includes a collection lens disposed so that light traveling from the object in the broad, flat flow is collimated by passing through the collection lens and travels along a collection path. A spectral dispersing element is disposed in the collection path so as to spectrally disperse the collimated light that has passed through the collection lens in a plane substantially orthogonal to a direction of relative movement between the object and the imaging system, producing spectrally dispersed light. (As noted above, the object or the imaging system or both can be in motion relative to the other and for the sake of simplicity, this relative movement is hereinafter referred to simply as xe2x80x9cthe movement.xe2x80x9d) An imaging lens is disposed to receive the spectrally dispersed light, producing an image from the spectrally dispersed light. Also included is a TDI detector disposed to receive the image produced by the imaging lens. As the movement occurs, the image of the object produced by the imaging lens moves from row to row across the TDI detector. The TDI detector produces an output signal that is indicative of at least one characteristic of the object, by integrating light from at least a portion of the object over time.
As a result of light collimation by the collection lens in this embodiment, all light emitted from a first point in the object travels in parallel rays. Light emitted from a second point in the object will also travel in parallel rays, but at a different angle relative to light from the first point. In this manner, spatial information in the object is transformed by the collection lens into angular information in the collection path. The spectral dispersing element acts on the collimated light such that different spectral components leave the spectral dispersing element at different angles, in a plane substantially orthogonal to the direction of the movement between the object and the imaging system. In this manner, both spatial and spectral information in the object are transformed into angular information. The imaging lens acts on the light from the dispersing element to transform different light angles into different positions on the detector. Spatial information is preserved by the system since light from the different positions in the object is projected to different positions on the detector, for both axes. In addition, light of different spectral composition that originates from the object is projected to different positions on the detector in an axis substantially orthogonal to the movement. In this manner, the spatial information from the object is preserved, while spectral information covering a large bandwidth is simultaneously collected at high resolution.
FIG. 16 further illustrates the simultaneous collection of spectral and spatial information by the present invention, when imaging male and female cells 200 and 208, respectively. Light of shorter wavelength, such as green laser scatter 212, will be focussed on the left side of the TDI detector. Light of slightly longer wavelength, such as yellow fluorescence 214 from a cell nucleus 202 or 210, will be laterally offset to the right. Light of still longer wavelengths, such as orange fluorescence 216 from an X-chromosome FISH probe and red fluorescence 218 from a Y-chromosome FISH probe, will be focused progressively farther to the right on the TDI detector. In this manner, different components of a cell that fluoresce at different wavelengths will be focused at different locations on the TDI detector, while preserving the spatial information of those components. Each component image may be broadened laterally due to the width of its associated fluorescence emission spectrum. However, this broadening can be corrected based upon a priori knowledge of the emission spectra. Deconvolution of the emission spectrum from the broadened component image will yield an undistorted component image. Further, since the spectral dispersion characteristics of the spectral dispersing element are known, the lateral offsets of the different color component images can be corrected to reconstruct an accurate image of the cell. Using this embodiment of the invention, high spatial resolution information can be collected simultaneously with high spectral resolution over several hundred nanometers of spectral bandwidth. It should clear to those skilled in the art that the present invention can be employed to enumerate numerous and multicolored FISH probes to simultaneously determine many characteristics from cells.
In the following disclosure, for all forms of the present invention where a prism is used as a spectral dispersing element, it can be replaced with a spectral dispersing component having characteristics that ensure no distortion or convolution of the image occurs due to the emission bandwidth, and as a result, a deconvolution is not needed to correct the image. Preferably, the spectral dispersing component that is used in place of a prism comprises a plurality of dichroic beam splitters, such as dichroic mirrors, which are arranged to reflect light within predefined bandwidths at predefined angles. Unlike a prism, where every wavelength leaves the prism at a different angle, all light within a predefined bandwidth incident on the dichroic beam splitter at a common angle leaves a given dichroic beam splitter at the same angle. Therefore, there is no convolution between the emission spectrum of the light leaving the object and the image of that object. When using such a spectral dispersing component, light of a first spectral bandwidth reflects off the first dichroic beam splitter at a predefined nominal angle. Light of a second spectral bandwidth is passed through the first dichroic beam splitter to the next dichroic beam splitter and is reflected therefrom at a different predefined nominal angle. Light of a third spectral bandwidth is passed through the first and second dichroic beam splitters to a third dichroic beam splitter and reflected therefrom at a third predefined nominal angle. The dichroic beam splitters are selected to cover the desired light spectrum with the appropriate spectral passbands. The angle of each dichroic beam splitter is set such that light reflected from it within the corresponding spectral bandwidth for the dichroic beam splitter is focused onto a different region of the detector. Since the present invention uses a narrow field angle in object space along the axis perpendicular to the axis of motion, many different spectral bandwidths can be simultaneously imaged onto a single detector. In this manner, each region on the detector may cover a different spectral bandwidth while collecting light over the same field angle in object space.
Depending on the amount of out-of-band rejection required, a bandpass filter is optionally placed in front of the detector. In one embodiment, the bandpass filter comprises a plurality of narrow spectral filters placed side-by-side to cover regions of the detector in correspondence with the spectral information to be imaged in those regions. Since the position of each spectral bandwidth region is predefined, and since the present invention maintains the spatial integrity of the object, a full color, high spectral resolution representation of the object is generated from the spectral information imaged onto the detector.
The use of a TDI detector in the present invention results in an extended imaging region along the axis of motion and a correspondingly long integration time. Several light sources can be simultaneously projected into the imaging region, increasing the amount of light incident upon objects therein. In addition, the combination of an extended imaging region and the orthogonal orientation of the spectral dispersion axis relative to the axis of the motion allows multiple objects to be imaged simultaneously. The long integration time and parallel image acquisition of this embodiment allows sensitive and consistent imaging performance to be combined with high throughput.
There are several alternative ways to provide light from the object. In one case, the light from the object comprises an unstimulated emission from the object, i.e., the object emits light without requiring a light source to stimulate the emission. In another embodiment, a light source is disposed to provide an incident light that illuminates the object. In this case, the object may scatter the incident light so that the light scattered from the object at least in part passes through the collection lens, or the incident light illuminating the object may stimulate the object to emit the light that passes through the collection lens. Further, the incident light may at least be partially absorbed by the object, so that the light passing through the collection lens does not include a portion of the light absorbed by the object. Finally, the incident light from the light source may be reflected from the object toward the collection lens. The light source or sources that are used preferably comprise at least one of a coherent light source, a non-coherent light source, a pulsed light source, and a continuous light source.
Spectral dispersion may be accomplished by many means, including a prism or grating. Further, although one preferred form of the invention employs a spectral dispersing element, the present invention is not limited to imaging the spectral dispersion of light. Alternatively, a dispersing element can be used to disperse light as a function of position, angle, polarization, phase, and other properties.
The object may be entrained within a fluid stream that moves the object past the collection lens, or alternatively, can be carried on a support, or simply move without the benefit of a support or flowing medium. Moreover, the present invention is not limited to the imaging of microscopic or small objects.
The TDI detector preferably responds to the image of the object by producing a signal that propagates across the TDI detector. Pixels of a typical TDI detector are arranged in rows and columns, and the signal propagates from row to row. However, the present invention is not limited to TDI detectors employing a rectilinear arrangement of pixels (e.g., a microchannel plate-based TDI detector). A propagation rate of the signal across the TDI detector can either be synchronized with a motion of the image of the object on the TDI detector as a result of the movement, or can be non-synchronized with the movement.
Other aspects of the present invention are directed to methods for imaging an object. These methods implement steps that are generally consistent with the imaging system discussed above.